The objective of the proposed studies is to determine if phosphoproteins or protein kinases, which are present in fetal liver and/or hepatocellular carcinoma, are induced in preneoplastic liver cells during promotion of chemical carcinogenesis in rat liver by choline deficiency. Nuclear and cytoplasmic phosphoproteins and protein kinases are known to be important in the regulation of eukaryotic gene expression and have been shown to differ between some normal and malignant tissues. Gamma-glutamyl transpeptidase positive (GGT+) hepatocytes (preneoplastic hepatocytes) will be isolated from livers of rats that have received a single dose of dietlylinitrosomine and have subsequently been fed a choline deficient diet. GGT+ hepatocytes will be separated from other cell types through centrifugation procedures and by use of a Becton Dickinson FACS4 cell sorter. Phosphoproteins and protein kinases from GGT+ hepatocytes will be compared with those from conrol hepatocytes and from fetal, regenerating and neoplastic liver. Phosphoproteins will be assayed by separation of nuclear and cytoplasmic32P labeled proteins on two dimensional polycrylamide gels. Protein kinase activity will be assayed quantitatively by the amount of incorporation of 32P from Gamma-labeled ATP into several substrates including nuclear nonhistone phosphoprotein from hepatoma. the presence of any qualitative differences between kinase activities will be judged from separation on 2D gels of the in vitro labeled products from the kinase reactions with the tumor nuclear nonhistone phosphoproteins. The proposed studies should provide valuable information regarding the role of protein kinases and phosphoproteins in the promotion of chemical carcinogenesis by choline deficiency and also, regarding the isolation of GGT+ hepatocytes.